Project Summary Trypanosoma brucei is the protozoan parasite responsible for Human African Trypanosomiasis (HAT), otherwise known as African Sleeping Sickness. There is an urgent need for new control strategies for this disease, as there is no efficacious vaccine. Furthermore, drugs used to treat the late stages of the disease are low in number, often toxic, and can be ineffective due to resistance. A thorough understanding of the parasite's biochemistry and its complex life cycle involving insects and mammals will potentially reveal novel targets for therapeutic intervention. Our recent studies indicate that different types of T. brucei RNAs contain the modified base 5-methylcytosine (5-MeC). Although there is a reasonable amount of literature on the role of 5-MeC in DNA molecules, there is little information regarding the role of 5-MeC in RNA molecules in all organisms including T. brucei. In addition, we have identified a family of putative cytosine RNA methyltransferases in T. brucei called TbCRMTs. Interestingly, two of the RNA methyltransferases, TbCRMT4 and TbCRMT5, are required for maximal parasite growth. Overall, our data leads to our hypothesis that RNA methylation and RNA methyltransferases are biologically significant in T. brucei, and RNA methyltransferases could represent drugs targets. This hypothesis will be directly tested in our proposed project. First, the entire 5-MeC methylome of RNA molecules in procyclic form and bloodstream form T. brucei parasites will be elucidated using RNA bisulfite sequencing (RBS-seq). We already have a small RNA methylome from our previous studies and thus we will focus on poly(A)+ RNA (which includes mRNA) and total RNA (which includes ribosomal RNA). Next, the role of the two critical TbCRMTs (4 and 5) in the described RNA methylation events will be evaluated. RBS-seq will be performed on RNA isolated from TbCRMT RNAi lines, and methylation levels will be compared to lines without an activated RNAi system. The effect of TbCRMTs on RNA levels and RNA processing will be determined, which will indicate how TbCRMTs and RNA methylation shape the transcriptome. In addition, the role of the two TbCRMTs in bloodstream form parasite growth will be evaluated, as current antitrypanosomal drugs target bloodstream form parasites. Finally, the two TbCRMTs will be expressed in bacteria, and recombinant TbCRMTs will be evaluated for in vitro cytosine RNA methyltransferase activity. Our studies will potentially improve control of HAT, as they will describe a new RNA metabolic pathway in T. brucei that is essential for parasite growth and survival.